Double dioxygenation of arachidonic acid by soybean lipoxygenase-1. Kinetics and regio-stereo specificities of the reaction steps.
The kinetic parameters of the first and second oxygenation of arachidonic acid by soybean lipoxygenase-1 were determined and found to be for the first step at pH 10.0, Km (arachidonic acid) = 8.5 +/- 0.5 microM; kcat = 225 +/- 7 s-1 and for the second step at pH 8.7 Km (15-HPETE) = 440 +/- microM; kcat = 25 +/- 1 s-1. In the second oxygenation for which 15-Ls-hydroperoxy 5-cis, 8-cis, 11-cis 13-trans-eicosatetraenoic acid is a substrate, two isomeric dihydroperoxy fatty acids are formed. After separation of the corresponding dihydroxy esters by high-performance liquid chromatography, they were identified by mass-spectrometry, 1H- and 13C-NMR spectroscopy as 8-DS, 15-LS-dihydroperoxy 5-cis, 9-trans, 11-cis, 13-trans-eicosatetraenoic acid and 5-DS, 15-LS-dihydroperoxy 6-trans, 8-cis, 11-cis, 13-trans-eicosatetraenoic acid. Independent evidence for the absolute configurations was obtained by capillary gas-liquid chromatography of diastereomeric R-(-)-2-butyl esters of the acetylated 2-hydroxy carboxylic acids produced by oxidative ozonolysis of the acetylated dihydroxy fatty acids. It is concluded that soybean lipoxygenase-1 produces hydroperoxides with predominantly the S-configuration irrespective of the position in the fatty acid which is oxygenated.
D001091:Arachidonate Lipoxygenases; D001095:Arachidonic Acids / Q000138:chemical synthesis; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008722:Methods; D010944:Plants / Q000201:enzymology*; D013025:Glycine max; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001095:Arachidonic Acids; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase